Nuska Tschammer - Academia.edu (original) (raw)

Papers by Nuska Tschammer

β-arrestin 2 pool titrations. HEK293T cells were cotransfected with fixed amounts of CXCR4-ElucC ... more β-arrestin 2 pool titrations. HEK293T cells were cotransfected with fixed amounts of CXCR4-ElucC and mock (a) or US28wt (b) cDNA and the DNA amount for ElucN-β-arrestin 2 was increased. After stimulation with vehicle or 100 nM CXCL12, luminescence was measured. ΔLuminesence was calculated by subtracting luminescence detected in vehicle stimulated cells from luminescence detected in cells stimulated with ligand for each transfection. Columns represent means ± SEM of at least two independent experiments each performed in quadruplicates. (TIF 130557 kb)

US27 does not alter surface expression of CXCR4 in HEK293T cells. HEK293T cells were cotransfecte... more US27 does not alter surface expression of CXCR4 in HEK293T cells. HEK293T cells were cotransfected with CXCR4, N-terminally tagged with FLAG, and mock or US27wt (a) Surface expression of CXCR4 was calculated as the signal ratio between permeabilized and non-permeabilized cells (reflected by FLAG-immunoreactivity) and normalized on the surface expression in CXCR4-only expressing cells. (b) The total expression of CXCR was calculated as a factor of FLAG-immunoreactivity in mock-transfected cells. (TIF 138263Â kb)

An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Journal of Proteins & Proteomics, 2018

While it’s very common for biologists and chemists to test whether or not two molecules interact ... more While it’s very common for biologists and chemists to test whether or not two molecules interact with each other, it’s much more useful to gather information on the nature of that interaction. How strong is it? How long will it last? What does that mean for its biological function? One way to answer these questions is to study affinity. Binding affinity is defined as the strength of the binding interaction between a single biomolecule to its binding partner, or ligand, and it can be quantifiably measured, providing information on whether or not molecules are interacting, as well as assigning a value to the affinity. When measuring binding affinity, there are several parameters to look at, but the dissociation constant (K d ), which defines the likelihood that an interaction between two molecules will break, is a very common measurement. The smaller the dissociation constant, the more tightly bound the ligand is, and the higher the affinity is between the two molecules. Keywords : Bi...

European Journal of Pharmaceutics and Biopharmaceutics, 2021

 Users may download and print one copy of any publication from the public portal for the purpose... more  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Breast Cancer: Basic and Clinical Research, 2019

We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-l... more We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The chemokine receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs...

Nature Methods, 2018

Surface plasmon resonance (SPR) is an optical methodology widely used to detect and quantify mole... more Surface plasmon resonance (SPR) is an optical methodology widely used to detect and quantify molecular interactions. SPR is considered the gold standard for quantification of protein interactions, but SPR assay optimization can be technically challenging, time-consuming and costly. Tycho™ NT.6 quickly analyzes different conditions typically tested during optimization of an SPR assay. The Tycho™ NT.6 system is simple to use and enables researchers to make better-educated decisions in developing and optimizing their binding-interaction assays. SPR and other biosensor-based analytical methods are standard tools used in academic and pharmaceutical research laboratories for the quantification of protein interactions. Although considered the gold standard by many scientists, the method can be technically challenging, time-consuming and costly to perform. The Tycho

European journal of medicinal chemistry, Jan 25, 2018

Based on the previously published pyrazolopyridine-based hit compound for which negative alloster... more Based on the previously published pyrazolopyridine-based hit compound for which negative allosteric modulation of both CXCR3 and CXCR4 receptors was disclosed, we designed, synthesized and biologically evaluated a set of novel, not only negative, but also positive allosteric modulators with preserved pyrazolopyridine core. Compound 9e is a dual negative modulator, inhibiting G protein activity of both receptors. For CXCR4 receptor para-substituted aromatic group of compounds distinguishes between negative and positive modulation. Para-methoxy substitution leads to functional antagonism, while para-chloro triggers agonism. Additionally, we discovered that chemotaxis is not completely correlated with G protein pathways. This is the first work in which we have on a series of compounds successfully demonstrated that it is possible to produce selective as well as dual-acting modulators of chemokine receptors, which is very promising for future research in the field of discovery of select...

Scientific reports, Jan 21, 2018

MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization... more MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques. To this end, we exploited the high-affinity interaction of tris-NTA with oligohistidine-tags, which are popular for purification, immobilization or detection of recombinant proteins. We used various DYE-tris-NTA conjugates to successfully label His-tagged proteins that were either purified or a component of cell lysate. The...

Molecular pharmacology, 2018

Our recent explorations of allosteric modulators with improved properties resulted in the identif... more Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 (-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)--[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[(-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([H]-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimidin-2-yl]ethyl}-2-[4-f...

Cell Communication and Signaling, 2016

Background: Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled r... more Background: Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish lifelong latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency. Results: We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection. Conclusions: We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.

Angewandte Chemie (International ed. in English), Jan 5, 2016

The chemokine receptor CXCR3 is a G protein-coupled receptor that conveys extracellular signals i... more The chemokine receptor CXCR3 is a G protein-coupled receptor that conveys extracellular signals into cells by changing its conformation upon ligand binding. We previously hypothesized that small-molecule allosteric CXCR3-agonists do not bind to the same allosteric binding pocket as 8-azaquinazolinone-based negative allosteric modulators. We have now performed molecular-dynamics (MD) simulations with metadynamics enhanced sampling on the CXCR3 system to refine structures and binding modes and to predict the CXCR3-binding affinities of the biased allosteric agonist FAUC1036 and the negative allosteric modulator RAMX3. We have identified two distinct binding sites; a "shallow" and a second "deeper" pocket to which the biased allosteric agonist FAUC1036 and negative allosteric modulator RAMX3 bind, respectively.

Scientific reports, Nov 8, 2016

G-protein coupled receptors (GPCRs) are important therapeutic targets since more than 40% of the ... more G-protein coupled receptors (GPCRs) are important therapeutic targets since more than 40% of the drugs on the market exert their action through these proteins. To decipher the molecular mechanisms of activation and signaling, GPCRs often need to be isolated and reconstituted from a detergent-solubilized state into a well-defined and controllable lipid model system. Several methods exist to reconstitute membrane proteins in lipid systems but usually the reconstitution success is tested at the end of the experiment and often by an additional and indirect method. Irrespective of the method used, the reconstitution process is often an intractable and time-consuming trial-and-error procedure. Herein, we present a method that allows directly monitoring the reconstitution of GPCRs in model planar lipid membranes. Plasmon waveguide resonance (PWR) allows following GPCR lipid reconstitution process without any labeling and with high sensitivity. Additionally, the method is ideal to probe the...

Scientific reports, Jan 12, 2016

G protein-coupled receptors (GPCRs), including dopamine receptors, represent a group of important... more G protein-coupled receptors (GPCRs), including dopamine receptors, represent a group of important pharmacological targets. An increased formation of dopamine receptor D2 homodimers has been suggested to be associated with the pathophysiology of schizophrenia. Selective labeling and ligand-induced modulation of dimerization may therefore allow the investigation of the pathophysiological role of these dimers. Using TIRF microscopy at the single molecule level, transient formation of homodimers of dopamine receptors in the membrane of stably transfected CHO cells has been observed. The equilibrium between dimers and monomers was modulated by the binding of ligands; whereas antagonists showed a ratio that was identical to that of unliganded receptors, agonist-bound D2 receptor-ligand complexes resulted in an increase in dimerization. Addition of bivalent D2 receptor ligands also resulted in a large increase in D2 receptor dimers. A physical interaction between the protomers was confirme...

Journal of medicinal chemistry, Jan 22, 2016

In this work we report a design, synthesis, and detailed functional characterization of unique st... more In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment. Concomitant structure-activity relationship studies indicated very steep structure-activity relationships, which steer the ligand bias between the β-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further d...

Protein Targeting Compounds, 2015

Residence time is a key factor responsible for efficacy of small molecule modulators of various t... more Residence time is a key factor responsible for efficacy of small molecule modulators of various types of receptors [1]. One way to prolong a receptor – ligand interaction is to introduce a mild Lewis acid moiety into ligand's structure. For example boronic acids were used as slowly reversible covalent proteasome inhibitors [2] and bortezomib – a peptide boronic acid – was approved in the USA for the treatment of several types of cancer. We employed this approach to design slowly reversible small molecule CXCR3 chemokine receptor modulators based on well characterized 8-azaquinazolinone scaffold. Previously published structure-activity relationship (SAR) and preliminary docking study suggested a spot for introduction of boronic acid moiety. According to this design we synthesized a set of boronic acids with different geometry and Lewis acid strength and tested them in binding and functional assays on CXCR3 receptors. Obtained results show that some boronic acid derivatives have e...

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intr... more Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gai protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gai-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy.